Aromatic compounds synthesized by the polyketide route are probably the most commonly occurring phenolic derivatives of natural origin, except the lignins. Little information is available on how microorganisms metabolize these compounds. Similarly, the catabolism of 2, 3-dihydroxybenzoates, formed by the shikimate pathway, is poorly understood; as is the role of 2, 3-dihydroxybenzoates as general converging ring fission catabolites. We propose to: (A) establish the main types of catabolic routes used by microorganisms to degrade these compounds; (B) study in detail the oxygenases involved in these transformations; (C) establish the relationships, if any, of the enzymes (or metabolic sequences) involved, to the well-described pathways of metabolism of other aromatic compounds; (D) provide further evidence for non-specific reaction sequences being responsible for the nutritional diversity of some microorganisms. The pathways of resorcinol and orcinol (3,5-dihydroxytoluene) metabolism have been partially elucidated in Ps. putida. The first enzyme of the reaction sequences resorcinol and orcinol hydroxylases are flavoproteins one of which we have crystallized. We propose to study these hydroxylases and the m-hydroxybenzoate hydroxylases from other pseudomonads in detail. Comparisons will be made of their physical properties and subunit structure, catalytic potential by study of the partial reactions, kinetics and specificity, stereospecificity, NIH shift, the alterations in substrate specificity induced by mutation and immunological homologies. We propose to examine the products accumulated by two mutants of Ps. putida blocked in the p-cymene pathway, forming 2, 3-dihydroxybenzoate. A study of the properties of acquisition mutants will also be initiated. The techniques used will include polarography, spectrophotometry, including the use of stopped flow devices, fluorimetry, and ORD-CD for the studies of the kinetic and catalytic properties of the enzymes.